阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录
Summary
了解脊椎动物的中枢神经系统的功能，需要从许多神经元的录音，因为皮层的功能所产生的，在神经元中的人口水平。在这里，我们描述了一个光学的方法来记录阈上的神经活动与单细胞和单穗分辨率，抖动随机存取扫描。这种方法记录体荧光钙信号与高时间分辨率从多达100个神经元。最大似然的算法deconvolves的基本阈上的神经活动，自体荧光钙信号。这种方法可以可靠地检测具有较高的检测效率和低误报率的尖峰，可以用来研究神经元群<em在体外</em>和<em在体内</em>。
Abstract
在脊椎动物的中枢神经系统的信息的信号往往是人口，而不是单个神经元的神经元。传播的阈上扣球活动涉及人群的神经元。直接寻址皮质功能的实证研究，因此需要高分辨率的神经元群体的录音。在这里，我们描述的光学方法和去卷积算法从多达100个神经元，单细胞和单穗分辨率记录神经活动。这种方法依赖于检测的瞬态与大脑皮质神经元的阈上电尖峰（动作电位）在体细胞内的钙离子浓度增加。高时间分辨率的光学记录的是，实现快速的随机访问扫描技术使用声光偏转器（AODs患者）1。双光子激发的钙敏感染料的查询结果在较高的空间分辨率在不透明的脑那朵起诉2。尖峰的荧光钙录音重构实现由最大似然方法。同时电和光学记录表明，我们的方法可以可靠地检测峰值（> 97％峰值检测效率），低利率的假阳性棘波检测（<0.003尖峰/秒），时间精度和高（约3 ms）3。可以用于这种秒杀检测的光学方法， 在体外和在体内的麻醉动物3,4的记录神经活动。
Protocol
1。光学装置（图1） 对于双光子激发的飞秒脉冲的红外脉冲激光系统被使用。高激光输出功率（在某些情况下，在890 nm波长大于2W）介绍了该系统的光学元件，以抵消巨大的损失。 由两个棱镜组成的甲prechirper系统赋予负的群速度色散（GVD）到激光脉冲之前的声光偏转器（季节AOD）补偿时间色散引入由AODs患者1。 两个季节AOD与大孔（10毫米的40倍水浸物镜NA 0.8）的激光?…
Discussion
间接地抖动随机存取扫描检测阈上扣球活动的增加与每穗在一个神经元的胞体在体细胞内的钙。胞内钙的增加，检测到由荧光钙染料。主要是从有限的信号 – 噪声比的钙荧光信号产生抖动的随机存取扫描的局限性。信号对噪声比是依次由光损伤，不允许使用高激发率限制。因为有限的信号 – 噪声比，穗的检测失败时，在某些神经元中，并也可能失败为持续和高频活动。例如，记录在体内时?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢兰迪Chitwood博士，批判性阅读的手稿。这项工作是支持的白厅基金会和斯隆基金会的赠款HJK。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Optical components are listed in order, starting from the laser | |||
| Titan:Sapphire Laser | Coherent Inc. | Chameleon Ultra 2 | High power output recommended (>2W at 900 nm) |
| Achromatic lens f = 30 mm | Thor labs | AC254-030-B | Anti-reflection (AR) coating for 650-1050 nm |
| Achromatic lens f = 100 mm | Thor labs | AC254-100-B | AR 650-1050 nm |
| lens f = 75 mm | Thor labs | LA1608-B | AR 650-1050 nm |
| lens f = 175 mm | Thor labs | LA1229-B | AR 650-1050 nm |
| Achromatic lens f = 300 mm | Thor labs | AC254-300-B | AR 650-1050 nm |
| Achromatic lens f = 100 mm | Thor labs | AC254-100-B | AR 650-1050 nm |
| Achromatic lens f = 100 mm | Thor labs | AC254-100-B | AR 650-1050 nm |
| Acousto-optical deflectors | Intraaction Corp | ATD 6510CD2 | |
| Reflective diffraction grating | Newport | 53-011R | 100 grooves/mm for AODs with 65 MHz bandwidth and scan angle of 45 mrad |
| 21.6 mm Brewster prisms | Lambda Research Optics Inc. | IBP21.6SF10 | |
| Colored Glass | Schott | BG-39 | |
| Dichroic mirror | Chroma Technology Corp | Z532RDC | |
| Photomultiplier modules | Hamamatsu | H9305-03 | |
| DAC-ADC board | National Instruments | PCI-6115 | |
| Oregon Green 488 Bapta-1 AM | Invitrogen | O-6807 |
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Tags
Optical RecordingSuprathreshold Neural ActivitySingle-cell ResolutionSingle-spike ResolutionPopulation Of NeuronsCortical FunctionIntracellular Somatic Calcium ConcentrationElectrical SpikesAction PotentialsTemporal ResolutionAcousto-optical DeflectorsTwo-photon ExcitationFluorescence Calcium RecordingsSpike Detection EfficiencyFalse Positive Spike DetectionTemporal Precision
Cite This Article
Ranganathan, G. N., Koester, H. J. Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution. J. Vis. Exp. (67), e4052, doi:10.3791/4052 (2012).